Vascular endothelial growth factor-A (VEGF-A) is an essential regulator of angiogenesis during embryogenesis and adulthood, and mediates pathological angiogenesis in many diseases, including cancer. VEGF-A signals through the receptor tyrosine kinases VEGFR1 and VEGFR2; the VEGFA gene encodes multiple VEGF-A isoforms, all of which bind VEGFR1 and VEGFR2, but differ in their capacity to bind to heparan sulfate proteoglycans (HSP) also present on target cells. HSP binding plays a critical role of VEGF-A signaling; mice engineered to express only a non-HSP binding isoform display defective microvessel branching and frequently die shortly after birth.
Vascular endothelial growth factor is a member of the PDGF family that is characterized by the presence of eight conserved cysteine residues and a cystine knot structure. Humans express alternately spliced isoforms of 121, 145, 165, 183, 189, and 206 amino acids in length. VEGF165 appears to be the most abundant and potent isoform, followed by VEGF121 and VEGF189. Isoforms other than VEGF121 contain basic heparin binding regions and are not freely diffusible.
Human VEGF165 shares 88% amino acid sequence identity with corresponding regions of mouse and rat, 96% with porcine, 95% with canine, and 93% with feline, equine and bovine VEGF, respectively. VEGF binds the type I transmembrane receptor tyrosine kinases VEGF R1 (also called Flt1) and VEGF R2 (Flk1/KDR) on endothelial cells. Although VEGF affinity is highest for binding to VEGF R1, VEGF R2 appears to be the primary mediator of VEGF angiogenic activity.
Within the carboxyl terminal domain of the VEGF165 isoform, amino acids R123, R124, and R159 are reportedly critical for HSP binding. However, it is noted that replacement of these amino acids with alanine causes reduced binding of VEGF 165 to VEGFR1 but does not affect binding of VEGF 165 to VEGFR2. Importantly, in an in vitro assay which assesses angiogenic activity, alanine substitution mutants of native VEGF showed the same ability to enhance angiogenesis as native VEGF. Alanine substituted mutants also showed reduced affinity for heparin, heparan sulfate proteoglycans, and for the VEGFR2 coreceptor, NRP1. VEGFR2 has been identified as the key signaling receptor that mediates the proliferative and migratory effects of VEGF. Alanine mutants were found to possess an angiogenic activity as potent as that of native VEGF154 (mouse), indicating that VEGF carboxyl-terminal domain is not directly involved in VEGFR2 binding.
The critical role of VEGF signaling in many human cancers, particularly as a driver of tumor growth and metastasis, for example, has made VEGF and its receptors important anti-cancer drug targets. VEGF signaling blockade has been achieved using VEGFR ATP binding site antagonists and VEGF neutralizing antibodies; each of these approaches has features as well as limitations. Neutralizing antibodies are highly selective for their intended target, e.g. VEGF, and have few, if any, off-target effects. ATP binding antagonists, in contrast, are often cheaper to manufacture than antibodies, but are less selective for their intended target and off-target toxicities are frequently associated with their use. Biological antagonists derived from the native protein ligand, e.g. VEGF, can be more cost-effective to produce than larger antibody molecules, yet possess a comparable level of target selectivity. A receptor antagonist would be desirable because a receptor antagonist would bind to the receptor, and would be likely to exert inhibitor action even where receptor activation is independent of VEGF binding. Bevacizumab (Avastin) is the major biological VEGF antagonist anti-cancer therapeutic approved so far by the US FDA. Bevacizumab is expensive to produce because it requires mammalian expression for manufacture, the most expensive recombinant expression system used in protein production. Aflibercept, a VEGFR fusion protein has US FDA approval for the treatment of colorectal cancer and wet macular degeneration. Among existing VEGF antagonists, Aflibercept has a unique target spectrum, although it too is manufactured using a mammalian cell expression system. It would be desirable to manufacture such an antagonist in a less expensive system, for example, via P. pastoris. 